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ELISA (enzyme-linked immunosorbent assay) is an immunoassay technology based on the specific binding of antigen and antibody. Its basic principle is to fix antigen or antibody of a specific concentration on the surface of a polystyrene microplate by physical adsorption, then add the sample to be tested, and use the degree of color development produced by the reaction between the enzyme label and the substrate to indirectly reflect the presence or absence or the amount of the antigen or antibody being tested.

As a type of immunolabeling technology (including immunofluorescence technology, immunoradiometric technology, immunoenzyme technology and immunocolloidal gold technology), ELISA technology has been widely used in scientific research and clinical experiments due to its characteristics of rapid, sensitive, qualitative or quantitative detection.

ELISA is one of the commonly used experimental methods in molecular biology, immunology and cell biology research, but in actual operation, people often encounter various experimental difficulties. For example, in ELISA experiments, a high blanking is one of the common problems, which not only affects the accuracy of the experimental results, but also may interfere with the reliability of the data. Therefore, it is particularly important to deeply understand the reasons for high blanking and take effective optimization measures.

High blanking refers to an abnormal increase in the absorbance value of the negative control or blank in the ELISA experiment. Normally, the absorbance value of the negative well should be less than 0.1. Too high a background value will interfere with the experimental results, reduce the accuracy and reliability of the data, and make it impossible to accurately assess the true content of the antigen or antibody in the sample.

Common causes include:

● Plate is not clean

Solution:

▼Wash thoroughly

Insufficient washing time will cause antibody residues, resulting in coloration of the negative control.

Try to extend the washing time, increase the number of washes, and add an appropriate amount of surfactant (such as Tween-20, 0.05%) to the washing solution.

When washing the plate at each step, ensure that the washing is thorough and tap the plate thoroughly until there is no obvious residual liquid in the well.

▼Avoid cross contamination

When discarding the washing solution or incubating antibodies, be careful to avoid cross contamination between wells.

Pipette tips should be used once as much as possible to prevent contamination caused by repeated use.

The filter paper used when tapping the plate should not be reused to avoid secondary contamination.

● Antibody or reagent concentration is too high

Reason: The concentration of antibody, enzyme-labeled secondary antibody or chromogenic substrate is too high, resulting in increased nonspecific reaction and increased background signal.

Solution:

▼Optimize antibody concentration: dilute the antibody to a suitable working concentration to avoid overdose.

▼Control reagent concentration: strictly adjust the concentration of enzyme-labeled secondary antibody and chromogenic substrate to prevent excessive concentration.

▼Perform gradient dilution experiment: gradually dilute through preliminary experiments to determine the optimal concentration of each reagent and reduce blanking interference.

● Incomplete blocking

Reason: The blocking solution (such as BSA) may cross-react with the antibody, resulting in an increase in background signal. If the blocking solution concentration is too low or the blocking time is too short, the blocking may not be complete, causing the antibody to bind non-specifically to the ELISA plate, thereby increasing the background value.

Solution:

▼Use a suitable blocking solution and adjust the concentration of the blocking solution.

▼The blocking time should be sufficient, generally 1-2 hours (room temperature) or overnight at 4°C.

▼Make sure to wash the plate wells after blocking to remove excess blocking solution.

●The color reaction is too long

Reason: Due to improper system optimization, the sample color reaction is weak. In order to compensate for this deficiency, color reaction time is extended. The long color reaction time leads to an increase in nonspecific reactions of the substrate.

Solution:

▼The detection system should be optimized, and the concentrations of the coating, detection antibody and substrate should be adjusted to ensure sensitive and specific reactions.

▼Shorten the color reaction time, generally no more than 15 minutes, to ensure the accuracy of the experimental results and avoid blanking interference.

● Reagent contamination or degradation

Reason: Contamination may occur during the preparation or storage of reagents and buffers, resulting in increased blanking.

Solution:

▼ Use freshly prepared reagents to avoid cross contamination.

▼ Use filtered solutions to ensure reagent purity.

▼ Check the expiration dates of antibodies, substrates, wash solutions, and buffers to avoid using deteriorated reagents.

● Temperature and environmental factors

Reason: Too high temperature or high humidity will lead to enhanced nonspecific reactions, resulting in increased blanking

Solution:

▼ Check the incubator to ensure that the incubation temperature is stable at 37°C and incubate according to the reaction time in the instructions.

▼ Avoid exposing the plate wells to high temperatures or strong light for a long time.

Instruments and equipment are the basis for the success of ELISA experiments. Commonly used related equipment includes pipettes, microplate readers, plate washers and incubators. All equipment needs to be regularly inspected and calibrated to ensure accuracy and precision. Welso provides high-quality equipment required for various ELISA experiments. Welcome to contact us for more information.